P1286: IMPAIRED ACTIVITY OF NATURAL KILLER CELLS IN DIFFUSE LARGE B-CELL LYMPHOMA MOST LIKELY ORIGINATES FROM LYMPHOMA-INDUCED MODULATION OF THEIR SURFACE RECEPTOR EXPRESSION
Authors: S. Ringelstein-Harlev, T. Azoulay, I. Slouzkey, M. Karmona, M. Filatov, G. Sarig
Journal: HemaSphere (2022)

Background: Natural killer (NK) cells play a role in cancer cell elimination; however, modifications in their activity induced by malignant cells can lead to solid and hematological cancer immune evasion. Diffuse large B-cell lymphoma (DLBCL) extensively infiltrates the lymphoid tissue, where environmental stimuli mediate the development of NK cell subpopulations. Aims: The aim of this study was to explore phenotypic and cytotoxic activity of DLBCL patient NK cells that could support lymphoma immune escape. Methods: Given that peripheral blood (PB) NK cells reflect lymph node subpopulations, CD56+ PB NK cells, derived from 26 DLBCL patients at staging, and from 13 healthy age-matched healthy controls (HC), were collected and analyzed for the surface expression of NK-cell specific C-type lectin receptors (CLRs), killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs) and other co-stimulatory receptors, using FACS. To assess NK cell activation, degranulation (evaluated by CD107a staining) and intracellular IFNg and TNFα production were examined following co-culture with the K562 malignant cell line. The percentage of CFSE stained target K562 cells that underwent necrosis/apoptosis upon co-incubation with NK cells was used to measure cytolytic activity of the latter cells. All statistical analyses were conducted with the Mann Whitney U test. Results: The percentage of NK cells among lymphocytes was comparable in patients and HC. As all the patient mature NK subsets were found to be CD56dim, all further experiments were conducted on this fraction only. The mean expression of the inhibitory CLR NKG2A was significantly higher in DLBCL than in HC samples (42%±5.1 vs. 27%±2.3; p<0.001). Conversely, the levels of activating CLRs NKG2D and CD161 were significantly lower in patients (15%±8 vs. 40.2%±4; p<0.01 and 47.8±7 vs. 82.7±5, respectively for both antigens; p=0.006). Patient NK cells demonstrated decreased levels of KIRs (CD158a and CD158b) compared to HC (16.2±5 vs 33.8±2.1; p<0.01 and 17.6±6 vs 32.4±3.1; p<0.01, respectively), despite being exclusively CD56dim, and as such expected to highly express KIRs. Degranulation was found to be lower in patient than in HC cells (CD107a of 2.8% vs. 14.6%; p<0.01), as was intracellular cytokine production [2.4% vs. 6.9% for TNFα (p=0.03) and 3.2% vs. 11.3% for IFNγ (p=0.04)]. This difference maintained upon IL-2 supplementation, which enhanced NK cell responses in both patient and HC cells. Non-receptor dependent activation with PMA/inomycin led to a marked and comparable increase in degranulation and cytokine production, in patient and HC cells. Dose-dependent lysis of target K562 cells was significantly lower with patient NK cells. At the highest effector:target cell ratio of 5:1, patient cells induced lysis of only 16.3% of target cells, whereas 24.2% of these cells were lysed following exposure to HC NK cells (p=0.001). IL-2 supplementation increased cytotoxicity, that still remained inferior in patient cells (20.7% vs. 28.1% target cell lysis; p=0.04). Summary/Conclusion: NK cells derived from DLBCL patients are found to display an aberrant surface receptor repertoire leading to receptor-dependent attenuated degranulation and cytokine production, along with diminished ability to promote lysis of malignant target cells. The demonstrated functional impairment of NK cells is likely to be driven by their exposure to the DLBCL environment and could promote lymphoma immune evasion.